Duchenne muscular dystrophy (DMD) is the most prevalent and severe form of human muscular dystrophy. DMD occurs with an incidence of 1 in 4000 male births. Onset of DMD is typically between 3 and 6 years of age with skeletal muscle weakness preferentially affecting the large proximal muscle groups. The disease is invariably progressive, leading to loss of ambulation by 11 to 13 years, and death typically in the 20's. Significant laboratory findings include grossly elevated serum CK-MM levels. Skeletal muscle biopsy samples reveal a dystrophic pattern of muscle degeneration and regeneration with fiber-size variation, increased central nuclei, and progressive interstitial fibrosis.
Becker muscular dystrophy (BMD) was long considered to be a potentially allelic disorder because of its clinical similarities to DMD and a common pattern of X-linked inheritance. The shared genetic basis for DMD and BMD was confirmed after the identification of the protein dystrophin; both DMD and BMD patients were shown to have dystrophin gene mutations. Typically, patients with DMD lack any detectable dystrophin expression in their skeletal muscles, and this is correlated with deletion mutations that disrupt the translational reading frame or point mutations that create stop codons. In contrast, muscle from patients with BMD contains mutated dystrophins having an altered size and/or reduced abundance secondary to deletion mutations that maintain the reading frame.
While clinical descriptions of DMD date back to the 1850's, over 100 years passed before evidence suggested that the muscle cell plasma membrane, or sarcolemma, is compromised in DMD muscle. The molecular basis for DMD and its associated sarcolemmal instability became more clear with landmark studies published in the mid-to-late 1980's which identified the gene encoding dystrophin as being defective in DMD (O'Brien and Kunkel, 2001). The DMD locus spans over 2.5 million bases distinguishing it as the largest gene in the human genome. The array of transcripts expressed from the DMD gene is complex due to the presence of multiple promoters and alternative splicing. The largest transcripts encode dystrophin, a four-domain protein with a predicted molecular weight of 427,000. Dystrophin is the predominant DMD transcript expressed in striated muscle. DMD gene mutations, deletions, or duplications most frequently result in a loss of dystrophin expression in muscle of patients afflicted with DMD. Based on its localization to the cytoplasmic face of the sarcolemma, and its sequence similarity with domains/motifs common to proteins of the actin-based cytoskeleton, dystrophin was hypothesized early on to play a mechanical role in anchoring the sarcolemma to the underlying cytoskeleton It has also been hypothesized that dystrophin plays a role in protecting the sarcolemma against stress imposed during muscle contraction or stretch.
Biochemical studies aimed at confirming the hypothesized structure and function of dystrophin revealed its tight association with a multi-subunit complex, the so-named dystrophin-glycoprotein complex. See FIG. 1, which is a schematic representation showing the sarcolemma and the interaction of dystrophin with the other elements of the dystrophin-glycoprotein complex. Through its cysteine-rich and C-terminal domains, dystrophin in striated muscle interacts with the integral membrane dystroglycan sub-complex and the sarcoglycan/sarcospan sub-complex, as well as the subsarcolemmal dystrobrevins and syntrophins (Cohn and Campbell, 2000; Blake et al., 2002). The N-terminal domain and a portion of middle rod domain of dystrophin act in concert to effect an extensive lateral association with actin filaments in vitro (Rybakova et al., 1996) and in vivo (Rybakova et al., 2000; Warner et al., 2002; Rybakova and Ervasti, 1997; Amann et al., 1998; Amann et al., 1999).
Utrophin is a widely expressed autosomal gene product with high sequence similarity to dystrophin (Tinsley et al., 1992). Utrophin is distributed throughout the sarcolemma in fetal and regenerating muscle, but is down-regulated in normal adult muscle and is restricted to the myotendinous and neuromuscular junctions (Blake et al., 1996). Because utrophin and dystrophin bind the same complement of proteins (Matsumura et al., 1992; Kramarcy et al., 1994; Winder et al., 1995), it was hypothesized that utrophin may be capable of compensating for dystrophin deficiency. Indeed, continued utrophin expression in adult mdx mice partially attenuates the phenotype associated with dystrophin deficiency. In short, mice lacking both dystrophin and utrophin exhibit a more severe phenotype similar to that seen in human DMD patients (Deconinck et al., 1997a; Grady et al., 1997). Moreover, transgenic overexpression of full-length utrophin completely rescued the dystrophic phenotype in mdx mice (Tinsley et al., 1998).
Methods to express and purify full-length utrophin using a baculovirus system has been demonstrated (Rybakova et al., 2002 and 2006). It has also been shown that purified recombinant utrophin is a soluble, rod-shaped monomer with the expected molecular weight of 400,000 Da. Recombinant utrophin-bound actin filaments display an affinity (Kd=0.2 μM) similar to that measured for purified dystrophin-glycoprotein complex (Rybakova et al., 2002). Recombinant utrophin-bound F-actin displays a stoichiometry of 1 utrophin per 14 actin monomers, which implies a more extensive lateral association with actin filaments than anticipated from studies with isolated fragments, but a less extensive lateral association than the 1 per 24 stoichiometry measured for purified recombinant dystrophin (Rybakova et al., 2006). Like the dystrophin-glycoprotein complex, recombinant utrophin protected actin filaments from forced depolymerization in a concentration-dependent manner that saturated at molar ratios equal to or greater than 1 utrophin per 14 actin monomers. Also different from purified dystrophin-glycoprotein complex, the binding of recombinant utrophin to actin filaments was completely insensitive to increasing ionic strength up to 0.8 M. These results (Rybakova et al., 2002) (Rybakova et al., 2006) indicate that dystrophin and utrophin both bind laterally alongside actin filaments through contributions by the spectrin-like repeats of the rod domain, but that the rod domain epitopes involved differ between the two proteins. Utrophin appears to bind laterally along actin filaments through a contribution of the first 10 acidic spectrin-like repeats (Rybakova et al., 2002) rather than a cluster of basic repeats as employed by dystrophin (Rybakova et al., 1996; Amann et al., 1998); (Rybakova et al., 2006).
Most viruses, including the human immunodeficiency viruses (HIV), encode proteins for regulating genome transcription. In HIV, the tat gene plays a role in driving the transcription of the HIV genetic code. The tat gene encodes a small nuclear protein of from 86 to 101 amino acids, depending upon the viral strain. Both the tat gene and its encoded protein, TAT, are known. The protein itself is designated TAT, for “transactivator protein.” The typical HIV-1 laboratory strains HXB2 and NL4-3 express an 86 amino acid-long TAT protein, while other HIV strains express a 101 amino acid-long TAT protein. See, for example, Kuppuswamy et al., 1989.
Despite all that is now known, and despite continuing efforts by many laboratories around the world (Gregorevic and Chamberlain, 2003), there is presently no cure or effective treatment to alleviate the devastating progression of DMD.